IMMUNOELECTROPHORESIS METHOD

Abstract: The present invention relates to a method of detecting in a test subject the presence of at least one hapten, one macromolecule, one pathogen or one part thereof that causes an immune response, as well as a method of detecting any immune response to the hapten, the macromolecule, the pathogen or at least one part thereof that causes an immune response. The invention also relates to the use of said method for screening test subjects for contamination with at least one pathogen or antigen, for screening for any infection with at least one pathogen and/or for screening for an allergy to at least one hapten or antigen.

Description: IMMUΝOELECTROPHORESIS METHOD The present invention relates to a method of detecting in a test subject the presence of at least one hapten, one macromolecule, one pathogen or one part thereof that causes an immune response, as well as a method of detecting any immune response to the hapten, the macromolecule, the pathogen or at least one part thereof that causes an immune response. The invention also relates to the use of said method for screening test subjects for contamination with at least one pathogen or antigen, for screening for any infection with at least one pathogen and/or for screening for an allergy to at least one hapten or antigen. The invention also relates to the use of the method for distinguishing contamination with a wild-type pathogen from that with modified pathogens, as well as for distinguishing any immune response after contamination with a wild-type pathogen from an immune response after contact with a modified vaccine. The method used most frequently at present for detecting specific antibodies to an antigen is ELISA (enzyme-linked immunosorbent assay). In ELISA, an antibody-antigen complex is detected by means of enzymatic colour reaction. Other frequently used techniques for detecting antigen-specific antibodies are RIA (radioimmunoassay) and IRMA (immuno radiometric assay) , where an antibody-antigen complex is detected by using radio¬ active labelling agents. The greatest disadvantages associated with work involving radioactivity are the costs and the risk of contamination of the environment and the individual carrying out the method. ELISA is not associated with the disadvantages mentioned in connection with radioactivity, and is more advantageous than RIA and IRMA in that the test is very sensitive and the costs when processing large numbers of samples are much lower. However, ELISA also has a number of disadvantages. ELISA can give many false-positive and false-negative assays because of the large number of vigorous washing steps required. There is only a relatively narrow margin within which the washing steps do not lead to false results; gentle washing always leads to false- positive readings, while vigorous washing removes immune complexes that have formed, so that they are not detected and false-negative results are obtained. The last-mentioned aspect occurs more frequently as the immune complexes become larger, for example in bacterial determinations where the complete microorganism is complexed. Furthermore, the ELISA technique requires many steps and is therefore fairly labour-intensive. In addition, ELISA indirectly reveals the presence of an immune complex by means of an enzymatic reaction. Moreover, it is not possible to detect an immune response of a test subject to pathogen A by the ELISA if an ELISA is being carried out with the aim of detecting any immune response to pathogen B. The simultaneous differentiation of disorders cannot be carried out effectively with ELISA techniques. However, it is possible, for example, to coat the wells in the microtiter plate used for an ELISA with a number of antigens specific to different pathogens, so that a number of antibodies characteristic of different infections with pathogens can be bound possibly from the sample, but in this case the sensitivity of the assay decreases considerably. Moreover, in this case it is only possible to demonstrate that the test subject shows an immune response to one of the pathogens, but it is impossible to determine to which pathogen. There is a need for a direct method of determining the antigens against which the test subject has formed antibodies, in particular in the case of infection with proteins characteristic of pathogens. For this purpose, proteins in a sample of a test subject will have to be separated from one another and/or immunocomplexes will have to be separated from the other non-complexed proteins. Electrophoresis is a frequently used technique for separating proteins in general. There are various types of electrophoresis, for example SDS gel electrophoresis, native gel electrophoresis and iso- electric focusing (IEF). In SDS gel electrophoresis the proteins are denatured; this is not the case with the last-mentioned two types. In the first two methods separation is effected via the size of the protein and in the last-mentioned type this is done on the basis of the isoelectric point. In view of the fact that antibodies electrophorese at the same point due to their large correspondence in terms of size, charge and mass and in view of the fact that direct detection of specific antibodies to a specific antigen in situ on the gel is impossible, because it is not possible to introduce the antigen in the gel at the same time as keeping the antibodies in place, the use of just electrophoresis for the specific detection of a certain antigen or antibody in a sample transferred to gel is impossible. Due to the fact that the component of the immune complex to be applied has to diffuse through the gel in order to reach the components of the sample and, if appropriate, to react therewith, and that of course the components of the sample also diffuse in the meantime, this makes it impossible to develop a reliable detection pattern. In order to solve this problem when applying electrophoresis in immunochemistry, the blotting techniques were developed. In these blotting techniques, protein-binding paper is brought into contact with a gel on which proteins have been separated. By application of pressure or electrophoresis, the proteins are subsequently transferred on to the paper, a fixed impression of the protein components being formed on the paper as they have separated on the gel. Specific detectable immune complexes can subsequently be formed in the same way as in ELISA and RIA by application of a specific antibody directed against the pathogen, part of a pathogen, hapten or protein to be detected, or an impression of the antibodies can be made on paper and the specific antigen can then be brought into contact with the latter and the immune complex which may result can then be rendered detectable. The blotting techniques also have disadvantages. The technique is slow, expensive, inefficient and insensitive. On blotting, the copying of the gel on to the paper, only part of the separated sample constituents is transferred on to the paper, so that the test is inefficient. In addition, the disadvantages reported for ELISA and RIA above apply in the same way. The existing techniques are insufficient for direct deter- mination of the antigens against which the test subject has formed antibodies. There is no test which shows in an efficient and simple manner that a test subject is contaminated and possibly infected and at the same time shows what has contaminated and possibly infected the test subject. In J. Clin. Chem.Clin. Biochem. Vol. 21, 1983, PP 8*11-844 Merlini G. et al. describe use of the immunosubtraction technique to identify the proteins in electrophoretically separated plasma. The technique consists of forcing the plasma through a layer of monospecific antibody purified by electrophoresis, thereby precipating the specific protein which is consequently absent from the subsequent electrophoretogram. The immunosubtraction technique consists of two steps: 1) electrophoretic purification of the gamma fraction of the antiserum and 2) electrophoresis of the sample through the purified antibodies. Antibody was applied with a microsyringe to an agarose gel and antisera were applied in alternate wells, leaving the adjacent wells free for the control, i.e. non-immunosubtracted, sera. Once absorbed, the antisera were subjected to electrophoresis at 20 V/cm for 4 min.. After this preliminary electrophoresis a strip of agarose gel, 1,5 cm wide, was removed from the deposition side of the plate because it is contaminated with the non-gamma fractions of the antisera and replaced with a new strip of the same gel. Subsequently the samples diluted with saline containing bromophenol-blue were placed both in the wells previously filled with the antisera, and in the adjacent wells which had been left free for comparative analysis. Immunosubtracted and non-immunosubtracted samples therefore migrated in parallel. The second electrophoretic run was carried out with inverted polarity then the samples were electrophoresed at 10 V/cm for the first 15 min. and at 20 V/cm for the following 30-40 min.. The current intensity was kept initially lower in order to facilitate the antigen antibody reaction. The disadvantages of this technique are numerous. With this technique it is impossible to detect antigens that migrate slower than the gammaglobulins because this would mean the antigens would never be able to overtake the gammaglobulins and form an immunocomplex with them. Also it is impossible to detect IgM with the method described by the Italians. It is only possible to detect IgG of the immunoglobulins. All the non-gamma fractions of sera have been cut away in the method according to Merlini et al.. The method according to Merlini et al. has the further disadvantages that two electrophoresis steps are required and that the electrophoresis must be carried out slowly, otherwise the antigen will run past the immunoglobulin without reacting with it and thereby causing false negative tests results. The method according to Merlini et al. is dependent on the migration speed of the substance which has to be detected in the sample. This substance must run faster through the gel than the binding partner that has been previously incorporated in the gel. Therefore, the method according to Merlini et al. requires a pre-analysis of which material runs faster in order to determine whether it can be detected or not. In particular, if one were interested in determining the presence of a specific pathogen such as a virus in a sample and one were to destroy the virus then the virus fragments would probably not migrate faster than the IgG fraction therefore be undetectable as causing immunocomplexes. In particular when one is interested in detecting an infection at an early stage one is interested in detecting the presence of IgM as this is produced in first instance of an infection whereas the IgG production at the beginning of the infection is very small and often not detectable. Therefore, the method of Metlini et al. could only be used to determine infection at a late stage of infection, in contrast to the subject method, which is capable of detecting IgM at an early stage of infection. In the US patent 5 228 960 published after the filing of the priority patent application of the subject invention, i.e. published on July 20, 1993 but filed prior to the priority application of that subject invention, i.e. July 17, 1992 a methodology is disclosed for capillary electrophoretic immunosubtraction of samples as a means of providing analysis of monoclonal gammopathies. In particular, the method disclosed is a capillary electrophoretic immunosubtraction method. Preferably of a sample comprising constituent parts to be separated, said method comprising the step of 1) separating a first aliquot of the sample into constituent parts by capillary electrophoretic techniques and detecting said parts, 2) admixing a second aliquot of said sample with at least one specific binding partner to a constituent of said sample, said specific binding partner capable of being substantially removed from said aliquot; 3) separating said aliquot into constituent parts by capillary electrophoretic techniques and detecting said parts; and 4) comparing the separated constituent parts of step 3) with the separated constituent parts of step 1). Preferably, the sample is a clinical sample and the constituent parts include immunoglobulins whereby the specific binding partner is an anti-immunoglobulin antibody. Most preferably, the specific binding partner is either insolubilized before the second aliquot is admixed therewith, or is capable of being insolubilized after admixture with the aliquot. In the method described in the U.S. Patent it is necessary to immunoprecipitate or remove any immunocomplexes from the sample as otherwise the capillaries will quickly be blocked by the large immunocomplexes. Surprisingly, a method has now been found with which the antigens against which a test subject, an experimental animal or a human being has formed antibodies can be determined directly by means of electrophoresis. The present invention relates to a method of detecting in a test subject the possible presence of at least one hapten, macromolecule, pathogen or one part thereof that causes an immune response, and/or of detecting any immune response to at least the hapten, macromolecule, pathogen or one part thereof that causes an immune response, in which a) a liquid sample of the test subject to be tested is taken or prepared, b) the sample is incubated with a stock solution, which stock solution comprises at least one characteristic antigen of the pathogen to be tested, of one part of the pathogen to be tested, of the macromolecule to be tested or of the hapten to be tested, or which stock solution comprises at least one antibody which is specific to the pathogen to be tested, for a part of the pathogen to be tested, for the macromolecule to be tested or for the hapten to be tested, c) the incubated mixture is applied, after incubation, to a substrate suitable for non-denaturing electrophoresis, d) the same stock solution that was used in step b) is applied to the same substrate as reference, e) the mixture of step c) and the stock solution of step d) are subjected to electrophoresis, the components of the incubation mixture and the stock solution being separated, f) the components of the incubation mixture and the stock solution are rendered detectable in one of the above-mentioned steps, and g) the signal patterns resulting after the electrophoresis of the incubation mixture and the stock solution are compared. The difference after electrophoresis between stock solution and sample due to the possible formation of immune complexes is analyzed. During the incubation step of the liquid sample with the stock solution, which stock solution comprises, for example, at least one specific antigen to be detected, any antibodies present in the sample which are specific to the antigen present in the stock solution can react to form the immune complex with their corresponding antigen. The method according to the invention can be used for detecting the presence of any type of immunoglobulin such as IgG, IgM, IgE, IgA. In particular the possibility of IgM detection is advantageous as early stages of infection can thus be positively diagnosed as infection. When the test subject has shown an immune response as reaction to contamination or inoculation with the specific antigen to be detected, the corresponding antibody specific to that antigen will be present in the serum sample of the test subject. The antibody will bind to the antigen present in the stock solution. ' Immune complexes will be formed. By forming the immune complex, certain molecular parameters of the antigen thus bound will change in relation to unbound antigen. This change in parameters, for example the isoelectric point and the molecular weight, can be observed in electrophoresis when the stock solution is employed as a reference in the electrophoresis in addition to the incubation mixture. Since it is intended with this method to monitor the complexes of proteins, in particular, of antibody-antigen, it is necessary to carry out the electrophoresis under non-denaturing conditions. Surprisingly, it has been found that the immune complexes formed do not denature or dissociate under the influence of gel consti¬ tuents such as glycerol or ampholines and that the intended object can therefore be achieved. An expert in the field of proteins knows which non-denaturing conditions are commonly used for protein separation in gel electrophoresis and can apply them readily when carrying out the method according to the invention. Although SDS-PAGE gives the best results for the blotting technique, it is unsuitable for the intended object because denaturation of proteins takes place. Native PAGE and IEF can be used in the method according to the subject invention. There are however noticeable differences between both techniques. The separation capability of IEF is much larger than the separation capability of native PAGE. Native PAGE electrophoresis is possible at a fixed pH, which is not possible with IEF. When using the subject invention one or the other of the techniques will be preferred. A person skilled in the art will appreciate which technique is preferred in which circumstances. Capillary electrophoresis is not useful in the subject method as immunocomplexes will block the capillaries. In all cases as the formed immunocomplex must remain in tact denaturing circumstances must be avoided. Noticeably the possible formation of immune complexes occurs prior to electrophoresis so that the reaction partners do not have to find eachother in the gel in contrast to existing methods, thereby eliminating a possible source of incorrect results such as occurs in the method of Merlini et al.. Prior to the subject invention a person skilled in the art would not have considered reacting the sample with a binding partner prior to electrophoresis as the resulting immunocomplexes were considered to be too large to enter a gel. Surprisingly it was found that a # acrylamide gel for example is quite capable of electrophoresis of immunocomplexes which have a size in the order of 500-600,000 Daltons. In particular it is even possible to detect situations where multiple antigens bind to antibody and also where multiple antibodies bind to an antigen. The general size of an immunoglobulin is 160,000 Daltons. In particular the difference to previous techniques is that in the current method the absence or presence of an immune complex is assessed whereas previously the immunocomplexes were discarded. The advantage of the present invention resides in the fact that the multiple washing steps and manipulations of the sample usually required with ELISA and the like are unnecessary. There are no false- positive or false-negative readings and the present method can also be carried out for large numbers of samples due to the easy procedure. Further advantage of the subject method lies in the fact that any type of antibody can be detected, not only an IgG but also an IgM or an IgA. It is also possible to use the method on samples comprising a little antibody and using excess antigen or for samples comprising a lot of antibody with addition of a little antigen. It is also possible to use the subject method when there is an equivalent amount of antigen to antibody. In short therefore the dosage of the amount of a specific binding partner is not relevant to the outcome of the test. All variants will give the desired results. Furthermore, the electrophoresis can occur in one simple step thereby simplifying the detection and thereby also lowering the costs. Furthermore, the technique no longer requires that the antigens have to be faster migratory elements than the specific binding partner. Nor is it necessary to carry out electrophoresis slowly in order to prevent false negatives as the reaction between this specific binding partner and the element to be detected occurs prior to electrophoresis thereby rendering the method independent of the voltage applied. In the method described by Merlini et al. a low voltage is necessary to avoid the false negatives. It is also possible with the subject method to detect early stages of infection in which generally speaking immunoglobulin G is produced in a very low amount and IgM is produced in a large amount, because the subject method allows detection also of IgM. It is possible with the present invention to screen for specific macromolecules or haptens, and not only for an "all-over" pathogen. A macromolecule can be understood to mean, inter alia, a protein or a polypeptide. Depending on the composition of the stock solution, it is also possible to screen for more than one disorder in a single operation. At the same time, it is possible with the method according to the invention to use precisely that pH range which is of interest with respect to a specific disorder. If IEF is employed as the electrophoresis step, electrophoresis can be carried out, for example, in a pH range of between 3 and 10 but, if screening is carried out for a specific contami¬ nation whose characteristic antigen electrophoreses at a pH of 6-7, electrophoresis can be carried out just within this pH range, so that a specific result can be obtained more quickly and more unambiguously. All the immune complexes and protein components of the gel can be rendered detectable by employing the usual colour reactions for proteins. The method according to the invention can also be carried out in such a way that each characteristic antigen or specific antibody of the stock solution in step b) is provided with a labelling agent. In this way, it is also possible to label different antigens or antibodies of the stock solution separately or in groups with different labels depending on what is to be detected. The present invention also relates to the use of the method according to the invention, which method is called electrophoretic profiling for the sake of clarity, for screening of a test subject for the presence of at least one pathogen or part thereof, or for screening of a test subject for an immune response in the case of infection or inoculation with at least one pathogen or part thereof. When used in this way, detection of a displacement and/or disappearance of at least one signal of the signal pattern of the incubation mixture in comparison with that of the reference after electrophoresis indicates contamination and possibly also infection with the applied pathogen, part of the pathogen that causes an immune response, macromolecule or hapten employed. The signal can be in the form of one or more bands or stripings on the gel or the peak of an electrophoretogram or can be the occurrence or absence of a large blotch or peak near or at the application site of the sample in the gel. In order to detect contamination with a specific pathogen, it is necessary to use the pathogen itself in the stock solution or to use at least one antigen characteristic of infection with the pathogen in the stock solution. If contamination with a number of different pathogens is to be detected by screening, the stock solution should comprise the corresponding pathogens or antigens thereof, which antigens lead to an immune response characteristic of an infection. However, if screening is used only to detect a contamination but not an immune response, it is possible to use a specific antibody for the pathogen to be detected in the stock solution and subsequently to monitor whether any complex formation has taken place in the incubation mixture according to the present electrophoretic profiling technique. Such complex formation can only take place if the antigen to be detected is present in the sample. Figure 1 illustrates use of the method according to the invention, i.e. electrophoretic profiling to determine infection with hepatitis B. The test was carried out in a 12# gel at 1500 Vh. Peak 3 is without antiserum, 4 with antiserum and 5 with lOx diluted antiserum. Figure 2 illustrates use of electrophoretic profiling to determine infection with IBR in a 4# gel at 1500 Vh. Peak 6 is without antigen. Peak 7 is with cow 1, peak 8 with cow 2 and peak 9 with cow 3- Quite clearly cows 1-3 are infected. Figure 3 illustrates the determination of the presence of antibodies to albumin using the method according to the invention on a 4% gel. In all figures the intensity of the band is given vertically and the location on the gel is expressed horizontally in mm. The invention also covers the use of the electrophoretic profiling technique according to the invention for screening for an allergy to at least one hapten or antigen, in which detection of complex formation in the incubation mixture according to the subject technique of electrophoretic profiling in comparison with that of the reference after electrophoresis indicates an allergy to the hapten or allergen employed. Another use of the present electrophoretic profiling technique, which is also interesting, is the use for distinguishing between contamination with a wild-type pathogen and immunisation with a modified vaccine, in which displacement and/or disappearance of signals with concomitant appearance of immunocomplexes in the gel or application site of the sample on the gel of all proteins characteristic for infection with wild-type pathogen indicates that an infection with a wild-type pathogen has taken place, and wherein the presence of at least one antigen, characteristic of an immune response and/or the absence of at least one antigen characteristic for immune reaction in the formed immunocomplexes, indicates that no antibodies have been formed against at least one antigen determining the immune response of the pathogen, which indicates that the immune response of the test subject is not directed against the wild-type pathogen but against the modified vaccine. In the present case, a modified vaccine is understood to mean a vaccine where either at least one protein is missing which causes a characteristic immune response on contamination with the wild-type pathogen, or a vaccine which comprises at least one protein mutated in such a way that binding with the antibody produced against the corresponding wild-type protein during infections no longer occurs. Using the electrophoretic profiling technique according to the invention, it is possible - to detect both the presence and absence of pathogens, - to detect the response of the body of a test subject to pathogens, parts of pathogens, antigens or haptens, as desired, and - to determine the state of health of the test subject as regards the chosen pathogens, parts of pathogens, proteins, haptens or antigens. The technique can be generally employed for diagnosing proteins present in body fluids of humans and animals. Such body fluids may be serum, blood, blood cells, urine, breast milk and saliva. The method according to the invention can be applied more specifically for - eradication programmes; demonstrating differences between natural immunity and immunity as a result of modified vaccines; - the preventative screening of populations, including company populations such as farm animals, for the presence of pathogens or the occurrence of immune responses to pathogens.

Claims: CLAIMS 1. Method of detecting the possible presence of at least one hapten, one macromolecule, one pathogen or one part thereof that causes an immune response in a test subject, of detecting an immune response to at least one hapten, one macromolecule, one pathogen or one part thereof that causes an immune response, in which a) a liquid sample of the test subject to be tested is taken or prepared, b) the sample is incubated with a stock solution, which stock solution comprises at least one characteristic antigen of the pathogen to be tested, of one part of the pathogen to be tested, of the protein to be tested or of the hapten to be tested, or which stock solution comprises at, least one antibody which is specific to the pathogen to be tested, for one part of the pathogen to be tested, for the macromolecule to be tested or for the hapten to be tested, c) the incubated mixture is applied, after incubation, to a substrate suitable for non-denaturing electrophoresis, d) the same stock solution that was used in step b) is applied to the same substrate as reference, e) the mixture of step c) and the stock solution of step d) are subjected to electrophoresis, the components of the incubation mixture and the stock solution being separated, f) the components of the incubation mixture and the stock solution are rendered detectable in one of the above-mentioned steps, and g) the signal patterns resulting after the electrophoresis of the incubation mixture and the stock solution in particular with regard to proteins and immune complexes are compared. 2. Method according to Claim 1, in which each characteristic antigen or specific antibody of the stock solution of b) is provided with a labelling agent. 3. Method according to Claim 1 or 2, in which native PAGE is carried out in step e) . 4. Method according to Claim 1 or 2, in which IEF is carried out in step e) . 5 * Method according to one of the preceding claims, in which the stock solution comprises at least the pathogen itself. 6. Use of a method according to one of the preceding claims for screening for the presence of or contamination and possibly also infection with at least one pathogen, in which detection of a displacement and/or disappearance of at least one signal of the signal pattern of the incubation mixture in comparison with that of the reference after electrophoresis in particular detection of a displacement and/or disappearance of at least one band or peak of the band or peak pattern and/or appearance of one or more bands or stripings in the gel due to the presence of immunocomplexes in the gel and/or presence of immune complexes at the application site indicates contamination with the pathogen employed. 7. Use of a method according to one of Claims 1-4 for screening for an allergy to at least one hapten or antigen, in which detection of a displacement of at least one signal of the signal pattern of the incubation mixture in comparison with that of the reference after electrophoresis in particular detection of a displacement and/or disappearance of at least one band or peak of the band or peak pattern and/or appearance of one or more bands or stripings in the gel due to the presence of immune complexes in the gel and/or presence of immune complexes at the application site indicates an allergy to the hapten or allergen employed. 8. Use of a method according to one or more of Claims 1-5. for distinguishing between infection with a wild-type pathogen and immunisation with a modified vaccine, displacement and/or disappearance of signals of the signal pattern, e.g. bands and simultaneous appearance of immune complexes in the gel or application site of all proteins characteristic for infection with wild-type pathogen indicates that infection with wild-type pathogen has taken place and wherein the presence of at least one antigen characteristic of immune reaction and/or the absence of at least one antigen characteristic for immune response in the formed immune complexes indicates that no antibodies have been formed against at least one antigen characteristic for the immune response of the pathogen, which indicates that the test subject is not infected with the wild-type pathogen but has had an immune response after contact with a modified vaccine.

Inventors: MOEN ALEXANDER ADRIANUS, VAN VOORST JAN JOHANNES, MOEN, ALEXANDER, ADRIANUS, VAN VOORST, JAN, JOHANNES
Applicants: MOEN ALEXANDER ADRIANUS, PROTEIOS B V, VOORST JAN JOHANNES VAN, MOEN, ALEXANDER, ADRIANUS, PROTEIOS B.V., VAN VOORST, JAN, JOHANNES
Application year: 1994
Main classification: G01N33
IPC codes: G01N27, G01N33